William N. Drohan

Further studies of blood infectivity in an experimental model of transmissible spongiform encephalopathy, with an explanation of why blood components do not transmit Creutzfeldt-Jakob disease in humans

BACKGROUND: Solid evidence from experimentally infected animals and fragmentary evidence from naturally infected humans indicate that blood may contain low levels of the infectious agent of Creutzfeldt‐Jakob disease (CJD), yet blood components have never been identified as a cause of CJD in humans.

Similar levels of infectivity in the blood of mice infected with human-derived vCJD and GSS strains of transmissible spongiform encephalopathy.

BACKGROUND: The possible transmission of variant CJD (vCJD) through blood transfusion or use of plasma‐derived products prompted this study comparing infectivity in murine models of vCJD and Gerstmann‐Sträussler‐Scheinker (GSS) disease, a non‐vCJD form of transmissible spongiform encephalopathy (TSE).

Expression and Purification of Functional Human α‐1‐Antitrypsin from Cultured Plant Cells

Human α‐1‐antitrypsin (AAT), the most abundant protease inhibitor found in the blood, was expressed in rice embryonic tissue suspension cell culture. This was accomplished by cloning the codon‐optimized AAT gene into a vector containing the rice RAmy3D promoter and its signal sequence. The synthetic gene incorporates codons synonymous with those found in highly expressed rice genes. Approximately 1000 stable transformed calli were produced by particle bombardment mediated transformation and were screened for high AAT expression using a porcine elastase inhibitory activity assay. The band shift assay also confirmed that rice‐derived AAT is functional regarding its binding capability to the elastase substrate. Time course studies were conducted to determine the optimum, postinduction expression levels from cell culture. AAT expression equivalent to 20% of the total secreted proteins was achieved, and a purification scheme was developed that yielded active AAT with purity greater than 95%. The potential applications of purified plant‐derived AAT for treatments of various AAT‐deficient diseases are discussed.

Utilization of an Epstein-Barr virus replicon as a eukaryotic expression vector.

Epstein-Barr virus (EBV) replicons which include the genetic element oriP and a functional gene for Epstein-Barr nuclear antigen (EBNA-1) can be maintained episomally in a variety of mammalian cell lines [Yates et al., Nature 313 (1985) 812-815]. We have assessed the application of an EBV replicon for foreign gene expression. Two cDNAs, human interferon-gamma (IFN-gamma) and the extracellular domain of the human epidermal growth factor receptor (EGF-Rex), cloned in an EBV replicon, were efficiently expressed and the protein was secreted into the extracellular media. Expression in human embryonic 293 cells was approximately ten-fold higher than in CV-1 cells. The expression of the human protein is dependent upon the orientation of the IFN-gamma transcriptional cassette relative to the other genetic elements within the vector.

Gene transfer efficiency during gestation and the influence of co-transfer of non-manipulated embryos on production of transgenic mice

Litter size of DNA microinjected zygotes is lower than for non-manipulated zygotes. The rate of embryonic and fetal survival in early, mid and late gestation was determined to assess whether DNA integration was responsible for embryonic losses. Also, the effect of including non-microinjected embryos with injected embryos on pregnancy rate and transgenic pup production was determined. In Experiment 1, one-cell embryos from immature CD-1 mice were microinjected with a whey acidic protein promoter-human protein C gene construct. One hour after microinjection embryos were transferred to pseudopregnant recipients (45 transfers of 30 embryos each). Fifteen recipients were sacrificed on day 4, 12 and 18 of gestation and the embryos/fetuses analysed for the transgene. The percentage of embryos or fetuses that were positive for the transgene was not significantly different at any day. However, the number of viable embryos at day 4 was significantly greater than fetuses on days 12 or 18. In addition, a high degree of mosaicism was observed in day 18 fetuses and placentae recovered. In Experiment 2, one-cell embryos from CD-1 mice were microinjected and co-transferred with non-manipulated embryos (C57BL/6). Pregnancy rate and the total number of pups born were improved by addition of non-injected embryos. However, the number of transgenic mice produced was similar whether non-injected embryos were included or not. There were 32.2% (15/46) transgenic pups when 0 non-injected embryos were transferred compared with 15.1% (13/86) transgenic pups when 4 or 8 non-injected embryos were added to the transfers. In summary, a high degree of embryonic and fetal mortality occurs among microinjected embryos. Furthermore, since the percentage of transgenesis did not change throughout pregnancy, DNA integration does not appear to account for all of the embryonic losses. other factor(s) related to the microinjection procedure may be involved in the embryonic and fetal failure of microinjected embryos. Addition of non-injected embryos, although it increased pregnancy rate and the number of pups born from microinjected embryos, actually decreased the number of transgenic pups obtained per pregnancy.

Inefficient processing of human protein C in the mouse mammary gland

Vitamin K-dependent plasma protein, human Protein C (HPC) has been expressed in transgenic mice, using a 4.2kb mouse whey acidic protein (WAP) promoter, 9.0 kb HPC gene and 0.4 kb 3′flanking sequences. Expression was mammary gland-specific and the recombinant human Protein C (rHPC) was detected in milk at concentrations of 0.1 to 0.7mg ml−1. SDS-PAGE revealed that the single, heavy and light chains of rHPC migrated with increased electrophoretic mobility, as compared to HPC. Enzymatic deglycosylation showed that these molecular weight disparities are in part due to differential glycosylation. The substantial increase observed in the amount of single chain protein, as well as the presence of the propeptide attached to 20–30% of rHPC, suggest that mouse mammary epithelial cells are not capable of efficient proteolytic processing of rHPC. TheKm of purified rHPC for the S-2366 synthetic substrate was similar to that of plasma-derived HPC, while the specific activity was about 42–77%. Amino acid sequence analyses and low anticoagulant activity of purified rHPC suggest that γ-carboxylation of rHPC is insufficient. These results show that proteolytic processing and γ-carboxylation can be limiting events in the overexpression of fully biologically active rHPC in the mouse mammary gland.

Transgenic pigs as bioreactors: a comparison of gamma-carboxylation of glutamic acid in recombinant human protein C and factor IX by the mammary gland

The mammary gland of transgenic livestock can be used as a bioreactor for producing complex therapeutic proteins. However, the capacity for making a given post-translational modification upon any given polypeptide is uncertain. For example, the efficiency of gamma-carboxylation of glutamic acid in the amino terminal regions of recombinant human protein C (rhPC) and recombinant human Factor IX (rhFIX) is different at similar expression levels. At an expression level of about 200 microg/ml in the milk of transgenic pigs, rhFIX is highly gamma-carboxylated as indicated by pro-coagulant activity and amino acid sequencing. However, only about 20-35% of rhPC has a native, gamma-carboxyglutamic acid-dependent conformation and anti-coagulant activity. Thus, this work provides an example of apparent differences in substrate specificity between two homologous proteins to the endogenous carboxylase of porcine mammary epithelium which leads to varying degrees of post-translational modification.

The Porcine Mammary Gland as a Bioreactor for Complex Proteinsa

The similar biological activity of rhPC and hPC indicates that porcine mammary gland can perform many of the processing reactions necessary for recombinant synthesis of complex human proteins and produce them at levels suitable for industrial bioreactor applications. The health of the transgenic pigs appeared unaffected by the expression of high levels of the heterologous protein. We suggest that one of the advantages of using the mammary gland as a bioreactor appears to be the high cell density relative to that of cell culture.

Development of an immunoaffinity process for factor IX purification.

Abstract. An immunoaffinity process based on monoclonal antibody (MAb) to factor IX (FIX) has been developed. Initially, vitamin‐K‐dependent proteins from cryoprecipitate‐poor plasma are isolated on DEAE‐Sephadex. The eluate is applied to an immunoaffinity column that utilizes a divalent metal‐ion‐dependent MAb directed against FIX. After washing the column with high salt in the presence of magnesium ion, the FIX is eluted using a citrate‐ or EDTA‐containing buffer. Coagulation assays and Western blots show no detectable amounts of any contaminating proteins. Purity of the FIX product is established using reduced and nonreduced Coomassie‐stanined SDS‐PAGE and HPLC. The N‐terminal 20 amino acids of the single peak of the HPLC were shown to be identical to those reported for FIX. The process shows no detectable leakage of monoclonal antibodies (MAb), efficient utilization of MAb, and provides yields greater than 95%. The use of solvent/detergent treatment as a potential viral inactivation method is incorporated in the process. Studies with tritiated Triton X‐100 indicate that the detergent can be washed out of the MAb column so that less than 1 ppm (total) Triton X‐100 coelutes with the FIX.

Phenotypic and genotypic stability of multiple lines of transgenic pigs expressing recombinant human protein C

The genotypic and phenotypic stability of four lines of transgenic pigs expressing recombinant human protein C in milk was examined. Two lines were established with a construct consisting of a 2.6 kb mouse WAP promoter and a 9.4 kb human protein C genomic DNA. Two lines were established with another construct consisting of a 4.1 kb mouse WAP promoter and a 9.4 kb human protein C genomic DNA. Genotypic stability was measured by transgene copy number transmission. Outbred offspring having a single transgene integration locus were established from a founder having three independent, multicopy loci. Phenotypic stability over multiple lactations was defined by the combination of recombinant human protein C expression levels and the isoform signature of recombinant human protein C in western blots. Both cDNA and genomic human protein C transgenes gave similar ranges of expression levels of about 100--1800 μg ml−1. Within a given outbred lineage having a single loci for the cDNA transgene, the expression levels ranged between 100--400 μg ml−1. Western blots of reduced recombinant protein C revealed that single chain content was not dependent on expression level and was consistent within each transgenic line, but varied between transgenic lines. This suggests that native swine genetics may play a role in selection of production herds with optimal post-translational proteolytic processing capability. Although swine are not conventional dairy livestock, it is agreed that the short generation times, multiple offspring per litter, stable paternal transmission of the transgene, and milk production capabilities of swine offer distinct advantages over conventional dairy livestock for the establishment of a herd producing a therapeutic recombinant protein